Fabrication of multi-well chips for spheroid cultures and implantable constructs through rapid prototyping techniques.

Three-dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi-well chips, which were validated using articular chondrocytes (ACs). Multi-well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type-II collagen deposition. Moreover, the use of our multi-well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical-grade fibrin to generate implantable multi-well constructs allowing for the precise distribution of multiple cell types. Multi-well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi-well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi-well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre-implantation samples. In conclusion, multi-well chips for the generation and culture of multiple cell spheroids can be fabricated by low-cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment.

Facile Fabrication of Three-Dimensional Hydrogel Film with Complex Tissue Morphology.

In this study, we proposed a simple and easy method for fabricating a three-dimensional (3D) structure that can recapitulate the morphology of a tissue surface and deliver biological molecules into complex-shaped target tissues. To fabricate the 3D hydrogel film structure, we utilized a direct tissue casting method that can recapitulate tissue structure in micro-/macroscale using polydimethylsiloxane (PDMS). A replica 3D negative mold was manufactured by a polyurethane acrylate (PUA)-based master mold. Then, we poured the catechol-conjugated alginate (ALG-C) solution into the mold and evaporated it to form a dried film, followed by crosslinking the film using calcium chloride. The ALG-C hydrogel film had a tensile modulus of 725.2 ± 123.4 kPa and maintained over 95% of initial weight after 1 week without significant degradation. The ALG-C film captured over 4.5 times as much macromolecule (FITC-dextran) compared to alginate film (ALG). The cardiomyoblast cells exhibited high cell viability over 95% on ALG-C film. Moreover, the ALG-C film had about 70% of surface-bound lentivirus (1% in ALG film), which finally exhibited much higher viral transfection efficiency of GFP protein to C2C12 cells on the film than ALG film. In conclusion, we demonstrated a 3D film structure of biofunctionalized hydrogel for substrate-mediated drug delivery, and this approach could be utilized to recapitulate the complex-shaped tissues.

Implantable and Injectable Biomaterial Scaffolds for Cancer Immunotherapy.

Cancer immunotherapy has become an emerging strategy recently producing durable immune responses in patients with varieties of malignant tumors. However, the main limitation for the broad application of immunotherapies still to reduce side effects by controlling and regulating the immune system. In order to improve both efficacy and safety, biomaterials have been applied to immunotherapies for the specific modulation of immune cells and the immunosuppressive tumor microenvironment. Recently, researchers have constantly developed biomaterials with new structures, properties and functions. This review provides the most recent advances in the delivery strategies of immunotherapies based on localized biomaterials, focusing on the implantable and injectable biomaterial scaffolds. Finally, the challenges and prospects of applying implantable and injectable biomaterial scaffolds in the development of future cancer immunotherapies are discussed.

A 3D Bioprinted Pseudo-Bone Drug Delivery Scaffold for Bone Tissue Engineering.

A 3D bioprinted pseudo-bone drug delivery scaffold was fabricated to display matrix strength, matrix resilience, as well as porous morphology of healthy human bone. Computer-aided design (CAD) software was employed for developing the 3D bioprinted scaffold. Further optimization of the scaffold was undertaken using MATLAB® software and artificial neural networks (ANN). Polymers employed for formulating the 3D scaffold comprised of polypropylene fumarate (PPF), free radical polymerized polyethylene glycol- polycaprolactone (PEG-PCL-PEG), and pluronic (PF127). Simvastatin was incorporated into the 3D bioprinted scaffolds to further promote bone healing and repair properties. The 3D bioprinted scaffold was characterized for its chemical, morphological, mechanical, and in vitro release kinetics for evaluation of its behavior for application as an implantable scaffold at the site of bone fracture. The ANN-optimized 3D bioprinted scaffold displayed significant properties as a controlled release platform, demonstrating drug release over 20 days. The 3D bioprinted scaffold further displayed formation as a pseudo-bone matrix, using a human clavicle bone model, induced with a butterfly fracture. The strength of the pseudo-bone matrix, evaluated for its matrix hardness (MH) and matrix resilience (MR), was evaluated to be as strong as original bone, having a 99% MH and 98% MR property, to healthy human clavicle bones.

In situ adjuvant therapy using a responsive doxorubicin-loaded fibrous scaffold after tumor resection.

As tumor microenvironment becoming more and more important in tumor study, the acid pH around or in solid tumors drew lots of attentions. And the progress of drug delivery systems made the responsive-release possible. This time, we fabricated a new-type composite electrospun poly (L-lactide) (PLLA) fibrous scaffolds, that blent with the mesoporous silica particles (MSNs). Further more, we used sodium bicarbonate (SB) as acid sensitive agent which was wrapped inside the MSNs. And doxorubicin (DOX) was also wrapped into MSNs in order to achieve a sustained release to inhibit tumors in mice, which mimicked the remnant breast cancer with surgery. In vitro experiments proved the characteristic of pH-responsive release of the composite fibrous scaffold. In vivo results showed that these composite fibers could induce obvious apoptosis and necrosis over 10 weeks. Further, the cancer-kill effects were also confirmed by the decreased level of Bcl-2 and TNF-α, while increased Bax and caspase-3 expression levels. Altogether, the results indicated that the composite drug delivery system as a local implantable scaffold could effectively kill cancer cells in a long term with pH-sensitivity after the tumor resection.